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Protistology 3 (2), 99-106 (2003) Protistology

Fine structure of nucleoli in the nasutum*

Bella P. Karajan1, Vladimir I. Popenko2 and Olga G. Leonova2

1 Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Avenue, 194064 St. Petersburg, Russia 2 Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street, 119991 Moscow, Russia

Summary

The macronuclear nucleoli of vegetative non-dividing cells of Didinium nasutum display inverted position of the main parts: the granular component is inside and the dense fibrillar one, in form of discrete bands, is mainly at the periphery. Before binary , the nucleoli are degranulated and their fibrillar bands scatter throughout the , to be segregated during division between the daughter cells, where they begin to re-form the granular parts. In young resting cysts small nucleoli consist of granular material only, larger nucleoli show a clear segregation of its fibrillar and granular elements. During conjugation, nucleoli of the old macronucleus or of its fragments become segregated into granular and fibrillar parts and the latter are largely eliminated. The nucleoli lose contact with the chromatin bodies. In the developing anlagen of the new macronuclei the first nucleoli appear as fibrous bodies and only later develop granular parts; simultaneously the number of nucleoli increases.

Key Words: macronucleus, nucleoli, cycle, cysts, Didinium nasutum

1995). The organization of rDNA in gymnostomes, Introduction including Didinium, is not known yet. The macronuclear nucleoli can fuse, separate, Macronuclear nucleoli are known to be very labile segregate into granular and fibrillar parts, or become structures. In all known cases, they are formed on degranulated depending on various environmental extrachromosomal (amplified) copies of ribosomal RNA factors, clonal growth phase, nutritional state, gene sequence, the so-called rDNA, which may be period, encystment, conjugation and so on (Raikov, monomeric, as in Glaucoma and all hypotrichs, dimeric 1982, 1995). These changes may occur on the repli- palindromic, as in or oligomeric as in cation level (i.e., via change of the copy number of the (Blackburn, 1982; Raikov, 1982, 1989, rDNA due to differential replication of the amplified extrachromosomal rDNA) or on the transcription level * In memory of Professor Igor Borisovich Raikov (i.e., due to differences in the rate of preribosomal RNA (30.12.1932-27.10.1998) synthesis), or else on the posttranscriptional level (i.e.,

© 2003 by Russia, Protistology 100 · Bella P. Karajan, Vladimir I. Popenko and Olga G. Leonova due to differences in the rate of rRNA processing and maturation of ribosomal particles). This paper aims to follow the fine structural changes of nucleoli at various stages of cell cycle of Didmium nasutum.

Material and methods The laboratory strain of Didinium nasutum used in the present work has been cultivated at room tempe- rature in lettuce medium and fed with Paramecium caudatum cultivated separately. The following fixation schedules were used: 1) 2% osmium tetroxyde in a 0.05 M sodium cacodylate buffer (pH 7.2), 30 min on ice. 2) A freshly prepared mixture of glutaraldehyde and osmium tetroxyde with the following final concen- trations: glutaraldehyde, 1%; osmium tetroxyde, 1%; phosphate buffer, pH 7.2, 0,05 M. Fix 30 min to 1 h on ice and in the darkness. The sections were routinely stained and studied with a JEM-100C electron microscope operated at 80 kv.

Results

VEGETATIVE CELL AND BINNARY FISSION The macronucleus of normally fed interphase cells contains numerous rather conspicuous nucleoli. The nucleoli show an inverted disposition of its main components: the dense fibrillar component mainly occurs in form of discrete bands at the surface of the nucleoli and around intranucleolar chromatin bodies, and the granular component lies inside the nucleoli. The nucleolar organizers in form of compact chromatin bodies either lie inside the nucleoli, surrounded by a ring of dense fibrillar bands, or at the surface, where they also contact bands of the dense fibrillar component (Fig. 1A). The latter bodies are indistinguishable from “free’’ chromatin bodies (see: Karadzhan and Raikov, 1977). Chromatin bodies, both intranucleolar and peripheral, often decondense forming fibrillar centres (NORs) as defined by Goessens (1974). When the macronucleus prepares to divide, it contracts from a horseshoe shape to a more or less rounded one and then begins stretching in a rod. During the stretching phase, numerous bundles of longitudinal assemble in the macronucleus. The nucleoli become largely degranulated and the dense fibrillar bands Fig. 1. Nucleoli in the interphase (А) and in a dividing become free and scatter throughout the macronucleus macronucleus (B, C). Abbreviations: c- chromatin during the contraction phase; their contacts with bodies, f - fibrillar component of the nucleolus, g - chromatin bodies are no more evident (Fig. 1B). The granular component of the nucleolus, inc - intra- general disposition of the nucleolar remnants is nucleolar chromatin body, n - nucleoli. Scale bars: reminiscent of a swirling movement described during pre- A, C - 0,5 µm, B - 1 µm. Protistology · 101

Fig. 2. Nucleoli in a young resting cyst. A - a general view of the macronucleus of a young cyst. The small granular nucleoli are designated by arrows. B - a relatively large nucleolus shown in A, at higher magnification. C - a small granular nucleolus (arrow) at higher magnification. Abbreviations: ln - a relatively large nucleolus (~ 800 nm in size) with segregated fibrillar and granular components, other abbreviations are the same as in Fig. 1. Scale bars: A - 1 µm, B, C - 0,2 µm. divisional contraction of the macronucleus in several considerably. Larger nucleoli show a clear segregation of (Raikov, 1982). fibrillar and granular component, but the segregation of During the stretching phase and the actual division the nucleolar component in young resting cysts obviously (pinching in two), the granular nucleolar component proceeds differently from that observed in the fragments begins to re-appear, but it is not yet organized into of old macronucleus during conjugation. The fibrillar regular nucleoli (Fig. 1C). The nucleolar material is component in this case is immersed in a granular mass of caught between bundles, the dense fibrillar the nucleolus (Fig. 2B). The small nucleoli consist of bands are mostly isolated and have the same longi- granular material only (Fig. 2C). Nucleolar organizers, tudinal orientation as microtubules (Fig. 1C). However, both inter- and perinucleolar, are not observed. the contact between chromatin bodies and fibrillar bands is re-established. All this argues for that the OLD MACRONUCLEUS DURING CONJUGATION biosynthetic activity of the nucleoli is resumed. During the first meiotic division of the (2-3 h after the start of conjugation), the old macronucleus CYSTS fragments into 20-30 pieces (Karadzhan and Raikov, Cells of Didinium nasutum can differentiate into 1979). Each piece usually contains several nucleoli. During resting cysts under unfavorable condition, such as the second meiotic division the peripheral position of the starvation, temperature stress and so on. We have bands of the dense fibrillar nucleolar component becomes investigated the young resting cysts, less than 16 h old, clearer than in vegetative cells; intranucleolar chromatin which formed spontaneously in a somewhat inhibited bodies disappear and the connection of the main culture. chromatin mass with the nucleoli is lost (Fig. 3A). This Number of nucleoli as well as the size of single can be considered as the onset of nucleolar segregation nucleoli in resting cysts reduce drastically (Fig. 2A). The into the fibrillar and the granular components. size of each nucleolus remains within the limits of 150 Somewhat later, usually still during the 2nd meiotic to 900 nm. The structure of nucleoli also changes division, the peripheral fibrillar bands contract and 102 · Bella P. Karajan, Vladimir I. Popenko and Olga G. Leonova coalesce into compact blocks (Fig. 3B). The rest of the bodies which depress the nucleolar surface (Fig. 3E). These nucleoli is granular. Nucleolar organizers, both intra- would enter the nucleoli and become the intranucleolar and perinucleolar, are absent. chromatin bodies. Simultaneously granular material During the first or the second synkaryon division begins to accumulate inside each nucleolus; additional the chromatin of the macronuclear fragments con- nucleoli are possibly also formed during this time, as their denses into a common spongy mass and at the same number grows. The fibrous bands always remain at the time the envelope of the fragments begins «to peel off» nucleolar surface (Fig. 3E). Sometimes such nucleoli fuse establishing direct contact of the macronuclear forming a large compound one. contents with the cytoplasm (Karadzhan and Raikov, 1979). The nucleoli remain segregated and devoid of Discussion NORs. They are located in holes in the chromatin mass and are always surrounded by a rim of struc- VEGETATIVE MACRONUCLEUS tureless nucleoplasm (Fig. 3C). Possibly the nucleoli give off a part of their granules to the cytoplasm. About In vegetative non-dividing macronuclei the the same time or a little later each macronuclear nucleolar morphology mainly depends on the nutri- fragment, together with some adjacent cytoplasm, tional state of the culture and/or on the phase of its becomes enclosed in a single-membraned vacuole. development. The results mainly concern Tetrahymena These vacuoles are likely to be autophagic. Later, where nucleoli fuse into larger aggregates during a during the early development of the macronuclear nutritional shift-down or simply in the stationary phase anlagen, when the fragments of old macronucleus (Raikov, 1982). This occurs also under the action of actually degene-rate, the nucleoli become completely certain drugs and other unfavorable conditions such as degranulated and their fibrillar blocks are hardly elevated temperature. Fused nucleoli are partly distinguishable inside the fused mass of pycnotic degranulated and show a strong inhibition of RNA chromatin. synthesis. Segregation of the nucleoli into fibrillar and granular parts under the action of certain drugs is also DEVELOPMENT OF THE NEW MACRONUCLEUS frequently observed (Caratero et al., 1983; Nilsson, 1985 ). After a nutritional shift-up (transfer to fresh The nucleoli appear in the anlagen of the new medium or refeeding) there is a burst of rDNA macronucleus for the first time at the «homogeneous» replication not associated with (apparently, (decondensed) stage of development of the anlagen that more copies of extrachromosomal rDNA are pro- occurs about 3 hours after separation of the conjugants duced): at the same time, rRNA synthesis is resumed (Karadzhan and Raikov, 1979). They are not very and the nucleoli return to normal morphology. Our numerous (10-20 per anlage) and extremely dense. study of Didinium concerns only normally fed cells. Almost all of them are ring-shaped, with a clearer Macronuclear nucleoli usually display a fibrillar centre and denser periphery (Fig. 3D). The centre is core and a granular cortex (Raikov, 1982, 1995). The often structured: it displays lighter and darker spots, whereas the periphery is similar to the material of the nucleolus-organizing region is then inside the fibrillar dense fibrillar bands of the nucleoli of vegetative core. However, in Didinium the position of the main macronuclei (Fig. 3D). The former possibly corres- components is inverted: they have a granular core and ponds to the NOR region, and the latter to the fibrillar a dense fibrillar cortex, and the NOR regions are mainly part of the nucleolus; no nucleolar granules exist at this at the periphery. A similar inverted position of components stage. These nucleolar primordia are usually surrounded occurs in (Vinnikova, 1974) and Nyctotherus with clear halos (shrinkage artefact?) and only (Vinnikova and Golikova, 1979). Moreover, the macro- incidentally connect with the general chromatin nuclear nucleoli of Didinium appear to be composite (a network with various threads (Fig. 3D). There are no product of fusion of several unit nucleoli) since they bodies of condensed chromatin in this network yet. have several NOR regions. Later, 4 to 6 hours after separation of the conjugants, the nucleoli enlarge and acquire a more typical CELL DIVISION structure. At the same time small bodies of condensed chromatin begin to form throughout the anlage; they It is well known that nucleoli diminish in size, are grow and become more and more abundant until they fragmented, or even disappear (at least, become invi- reach the morphology typical of the vegetative macro- sible with the light microscope) during division of the nucleus. The clear halos around the nucleoli diminish, and macronucleus in many ciliates. Often the nucleoli are the nucleoli come into contact with some chromatin degranulated (Raikov, 1982). In Didinium, the composite Protistology · 103

Fig. 3. Nucleoli in the old macronuclear fragments and in an anlage of a new macronucleus following conjugation. A, B - segregation of nucleolar material into granular and fibrillar components. C - a fragment of an old macronucleus, showing spongy chromatin mass, segregated nucleoli and ruptures of the nuclear envelope. D - first nucleoli with a clearer center and denser periphery in the anlage of a new macronucleus. E- a nucleolus in the anlage of a new macronucleus, 24 h after separation of the conjugants. Abbreviations are the same as in Fig. 1. Scale bars: A, B, D - 1 µm, C - 5 µm, E - 0,5 µm. 104 · Bella P. Karajan, Vladimir I. Popenko and Olga G. Leonova nucleoli are not only degranulated but fall into unit fibrillar of the dense fubrillar component. Segregation of nucleolar bands already during preparation to division, and during components is known to occur in cells of various organisms division itself the nucleolar granules begin to re-form. All when biosynthesis is lowered (see review: Bush and this indicates a temporary decrease of the biosynthetic Smetana, 1970). It also occurs during encystment of some activity of the nucleoli just prior to cell division. protozoans, e.g., Arcella (Raikov et al., 1989), and in the old macronucleus during conjugation in some ciliates, e.g., (Kovaleva et al., 1998). After that the nucleoli CYSTS in Didinium become largely degranu-lated, also like in It is well known that during encystment morpho- Blepharisma, possibly as a result of migration of nucleolar logy of nucleoli undergoes drastic changes (Raikov, granules to the cytoplasm through gaps in the nuclear 1982; Gutierrez et al., 1991, 1998). In some ciliates envelope of the macronuclear frag-ments (Karadzhan and nucleoli in resting cysts can fuse (Frenkel, 1992; Palacios Raikov, 1979). et al., 1995) or disappear (Foissner and Foissner, 1987; Final degeneration of the old macronucleus or its Adl and Berger, 1997). A segregation of fibrillar and fragments usually involves destruction of its envelope granular components, and subsequent disappearance of and enclosure in autophagic vacuoles. This occurs, the latter was observed in cysts of Tillina magna (Frenkel, e.g., in (Skarlato, 1978), Dileptus (Vinnikova, 1989, 1992). In cysts of Hypotrichida macronuclei 1974), and Tetrahymena (Weiske-Benner and Eckert, become small, compact and homogeneous (Grimes, 1987). Simultaneously, the RNA synthesis, which has 1973; Walker et al., 1980; Jareno, 1985). For these been active before this moment, is arrested. an appearance of bundles of microtubules at the early stages of encystment is typical. Nucleoli are bound to ANLAGEN OF THE NEW MACRONUCLEUS these microtubules. In and some other ciliates, the amount of In the developing anlagen of the new macronucleus preribosomal granules in the young cysts increases in development of nucleoli proceeds in some-what different comparison with precyst stage (Chessa et al., 1983). The ways in various ciliates (see reviews: Raikov, 1982, 1995). granular component is conspicuous in nucleoli of Colpoda In D. nasutum the first nucleoli appear in the form of steini (Frenkel, 1987), Dileptus visscheri (Kink, 1973). The fibrous bodies about 3-4 hours after separa-tion of the presence of granular component in the nucleoli and conjugant. In Paramecium aurelia, the first nucleoli clustering of preribosomal granules into 100-200 nm RNP appear in the anlagen earlier, about 30 min after particles was observed in resting cysts of Bursaria truncatella differentiation of the latter (Jurand et al., 1964; Berger, (Popenko et al., 1998) and B. ovata (Samoshkin and 1973). In most hypotrichs the nucleoli are formed much Sergejeva, 1995). The bundles of microtubules in later, only after destruction of the polytene chromosomes macronuclei of these species were not observed. (Ammermann, 1971; Murti, 1973; Skarlato, 1978; etc.). More or less similar changes of nucleoli occur also Late formation of the nucleoli in macronuclear anlagen in young resting cysts of Didinium nasutum. Nucleoli was observed also in Stentor (Skarlato, 1978), Blepha- decrease in size. Small nucleoli consist of granular risma (Kovaleva et. al., 1998) and some other ciliates. material only. Intranucleolar and peripheral chromatin bodies are absent. All these structural changes correlate Conclusion well with transition of the cell to the resting stage. The presence of aggregates of preribosomal Results presented in this paper show that the granules in cysts can be explained by accumulation of structure of nucleoli undergoes drastic changes at various rRNP for subsequent processes of cyst formation and/ stages of life cycle in D. nasutum. Fibro-granular nucleoli, or excystment in the future (Chessa et al., 1983; containing intra- and perichromatin bodies in the Frenkel, 1987). It is possible, however, that structural macronucleus at interphase, can degranulate during changes observed in early cysts can be explained by a binary fission, segregate in resting cysts and in fragments stronger preribosomal RNA suppression in com- of old macronucleus, appear in autophagic vacuoles and parison with that of rRNA synthesis (Eckert and re-form in the macronuclear anlagen after conjugation. Franke, 1975). The structure of nucleoli may be an indicator of the level of biosynthetic activity of the cell, indicating changes in the rRNA metabolism. OLD MACRONUCLEUS AT CONJUGATION The first changes that occur in nucleoli in the Acknowledgements fragments of the old macronucleus of Didinium is their Part of this work was carried out with financial progressive segregation and subsequent compactization support of the Russian Foundation for Basic Research Protistology · 105

(project No. 02-04-49064). The authors are very Gutierrez J.C., Martin-Gonzalez A. and Matsusaka T. grateful to Aleksey Ibragimov (St. Petersburg) for 1991. Towards a generalized model of encystment technical assistance in preparing the manuscript. (cryptobiosis) in ciliates: a review and hypothesis. BioSystems. 24, 17-24. References Gutierrez J.C., Martin-Gonzalez A. and Callejas S. 1998. Nuclear changes, macronuclear chromatin Adl S.M. and Berger J.D. 1997. Timing of life cycle reorganization and DNA modifications during morphogenesis in synchronous samples of Ster- ciliate encystment. Europ. J. Protistol. 34, 97-103. kiella histriomuscorum. I. The vegetative cell cycle. Jareno M.A. 1985. Etude ultrastructurale de l’enkys- Europ. J. Protistol. 33, 99-109. tement et du dekystement chez Onychodroinus Ammermann D. 1971. Morphology and development acuminalus (Ciliata, Hypotrichida). Protistologica. of the macronuclei of the ciliates 21, 313-321. mytilus and aediculatus. Chromosoma. 33, Jurand A., Beale G. H. and Young M.R. 1964. Studies 209-238. on the macronucleus of Paramecium aurelia. II. Berger J.D. 1973. Nuclear differentiation and nucleic Development of macronuclear anlage. J. Proto- acid synthesis in well-fed exconjugant of Para- zool. 11, 491-497. mecium aurelia. Chromosoma. 42, 247-268. Karadzhan B.P. and Raikov I.B. 1977. Fine structure Blackburn E.H. 1982. Characterization and species of the nuclear apparatus of Didinium nasutum differences of rDNA: protozoans. In: The Cell (Ciliophora, Gymnostata) in interphase and during Nucleus, Vol. 10. Acad. Press, N.Y.- London. pp. binary fussion. Protistologica. 13, 15-29. 145-170. Karadzhan B.P. and Raikov I.B. 1979. Ultrastructural Bush H. and Smetana K. 1970. The nucleolus. Acad. and radiographic investigation of the resorption Press, N.Y.- London. of the old macronucleus following conjugation Caratero C., Bes J.C., Caratero A. and Planel H. 1983. in Didinium nasutum. Protistologica. 15, 507- Etude ultrastructurale du nucleole et de la nucleo- 519. logenese chez Tetrahymena pyriformis. Protis- Kink J. 1973. The organization of fibrillar structures in tologica. 19, 177-186. the trophic and encysted Dileptus visscheri (Ciliata, Chessa M.G., Delmonte-Corrado M.U. and Ramoino Rabdophorina). Acta Protozool. 12, 173-194. P. 1983. Studio della fine struttura del macronucleo Kovaleva V.G., Raikov I.B. and Miyake A. 1998. di Colpoda cucullus. II variare del sistema nucleo- Nuclear fine structure at conjugation of the ciliate lare nel corso del ciclo di sviluppo. Atti Accad. Lig. Blepharisma japonicum and the alternative way of Sci. Lett. 40, 1-15. macronuclear development. Tsitologiya. 40, 190- Eckert W.A. and Franke W.W. 1975. Changes in fine 199 (in Russian with English summary). structure and composition of macronuclei of Murti K.G. 1973. Electron-microscopic observation Tetrahymena pyrifosmis induced by drugs interfering on the macronuclear development of Stylonychia with RNA synthesis and processing. Cyto-bilologie. mytilus and Tetrahymena pyriformis (Ciliophora, 11, 392-418. ). J. Cell Sci. 13, 479-509. Foissner I. and Foissner W. 1987. The fine structure of Nilsson J. R. 1985. Dose - and time-dependent effects the resting cysts of Kahliela siplex (Ciliata, of actinomycin D on Tetrahymena, with special Hypotrichida). Zool. Anz. 218, 65-74 reference to nucleolar changes. Kongress Dansk. Frenkel M.A. 1987. Fine structure of the resting cysts Videnskab. Selskab. Biol. Skifter 24, 3-16. of the ciliate Colpoda steini. Tsitologiya. 29, 131- Palacios G., Martin-Gonzalez A. and Gutierrez J.C. 136 (in Russian with English summary). 1995. Macronuclear chromatin study in vegetative Frenkel M.A. 1989. Nucleolar apparatus of the ciliate cells and resting cysts of Colpoda inflata: chromatin Tillina magna at encystment. Tsitologiya. 31, 594- spreading and standard transmission electron 596 (in Russian with English summary). microscopy. Second European Congress of Pro- Frenkel M.A. 1992. Fine structure of the macronucleus tistology and Eighth European Conference on in the resting cysts of the ciliate Tillina magna. Arch. Ciliate Biology. Clermont-Ferrand (France). p.43. Protistenkd. 141, 27-40. Popenko V.I., Cherny N.E., Ivanova J.L. and Yakovleva Goessens G. 1974. Nucleolar structure. Int. Rev. Cytol. M.G. 1998. Ultrastructure of macronucleus in the 87, 107-158. resting cysts of the ciliate Bursaria truncatella. Eur. Grimes G.W. 1973. Differentiation during encystment J. Protistol. 34, 18-28. and excystment in Oxytricha fallax. J. Protozool. Raikov I.B. 1982. The protozoan nucleus. Morphology 20, 92-104. and evolution. Springer-Verlag, Wien-New York. 106 · Bella P. Karajan, Vladimir I. Popenko and Olga G. Leonova

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Address for correspondence: Bella P. Karajan. Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Avenue, 194064 St. Petersburg, Russia. E-mail: [email protected]

Editorial responsibility: Sergei Fokin